Scope and Limitations of the Nicking Enzyme Amplification Reaction for the Synthesis of Base-Modified Oligonucleotides and Primers for PCR

0394750 - ÚOCHB 2014 RIV US eng J - Článek v odborném periodiku
Ménová, Petra - Raindlová, Veronika - Hocek, Michal
Scope and Limitations of the Nicking Enzyme Amplification Reaction for the Synthesis of Base-Modified Oligonucleotides and Primers for PCR.
Bioconjugate Chemistry. Roč. 24, č. 6 (2013), s. 1081-1093. ISSN 1043-1802. E-ISSN 1520-4812
Grant CEP: GA ČR GA203/09/0317
Institucionální podpora: RVO:61388963
Klíčová slova: isothermal DNA amplification * cross-coupling reactions * nucleoside triphosphates * polymerase incorporation * functionalized DNA * nucleic-acids
Kód oboru RIV: CC - Organická chemie
Impakt faktor: 4.821, rok: 2013

Enzymatic synthesis of short (10-22 nt) base-modified oligonucleotides (ONs) was developed by nicking enzyme amplification reaction (NEAR) using Vent(exo-) polymerase, Nt.BstNBI nicking endonuclease, and a modified deoxyribonucleoside triphosphate (dNTP) derivative. The scope and limitations of the methodology in terms of different nucleobases, length, sequences, and modifications has been thoroughly studied. The methodology including isolation of the modified ONs was scaled up to nanomolar amounts and the modified ONs were successfully used as primers in primer extension and PCR Two simple and efficient methods for fluorescent labeling of the PCR products were developed, based either on direct fluorescent labeling of primers or on NEAR synthesis of ethynylated primers, PCR, and final click labeling with fluorescent azides.
Trvalý link: http://hdl.handle.net/11104/0223103